C'! of S. pneumoniae" 1R01AI01808903953-051-OA1IAI "Impact of Antibiotics and Vaccines on the in vivo Evolution CO) "Impact of Antibiotics and Vaccines on the in vivo Evolution 3.0 m Streptococcus pneumoniae has remained one of the major causative agents of potentialylifethreattenininghumandiiseaseininourerra.. Nevertheless, introduction of antibiotics (peniciillliin)and- more recently - ttheconjjugate vaccine provoked majjor evolutionary changes in the structure of natural populations of this pathogen. The purpose of this research proposal is to combine the power of whole genome sequencing (WGS) and epidemiology to obtain insights into the mechanisms by which antibiotic resistant and non-vaccine type pneumococcal lineages emerged in the in vivo environment. The studies will have three foci of concentration. Proiect I. Three highly penicillin resistant pneumococcal (PRPn) clones: ST-I, ST-2 and ST-3, each with a unique sequence type (MLST) and capsular type have achieved pandemic spread and appear to remain stable over long times and distant geographic sites of isolation. Such genetic stability in a highly recombinogenic pathogen is unusual and it contrasts with the well documented diversity of penicillin susceptible pneumococci. The purpose of this project is to use whole genome sequencing (WGS) to better document and understand the nature and mechanisms of genetic stability in these three PenR clones. Proiect II. Pneumococcal strains expressing the non-vaccine type (NVT) capsular polysaccharides, such as 1 IA, 6A or 1 9A have been identified as minority components of the nasopharyngeal flora of children attending Day Care Centers - even before the introduction of the conjugate vaccine. WGS will be used to test if the emergence of these strains from minority to majority status in the nasopharynx and from is accompanied by changes in genetic makeup Because the pandemic penicillin-resistant strains show much less genomic variation over time and distance than the nonclonal strains, structure from the nonclonal types we will investigate these strains to see if they are less recombinogenic and/or limited with respect to donor DNA source as opposed to being less capable or less likely to form mixed strain biofilm structures with penicillin-sensitive strains thus resulting in lower rates of inter-strain transformation and evolution. =(D f/1 colonizers to disease causing strains - is accompanied by changes in genetic makeup rates of inter-strain transformation and evolution. mixed strain biofilm structures with penicillin-sensitive strains thus resulting in lower respect to donor DNA source as opposed to being less capable or less likely to form cell wall structures are profoundly different in structure from the nonclonal types we will genomic variation over time and distance than the nonclonal strains, and because their investigate these strains to see if they are less recombinogenic and/or limited with Proiect Ill. Because the pandemic penicillin-resistant strains show much less r-. cell wall structures are profoundly different in colonizers to disease causing strains - three Pen capsular polysaccharides, components of the nasopharyngeal flora of children attending Day Care Centers - emergence of these strains from minority to majority status in the nasopharynx and from before the introduction of the conjugate vaccine. is unusual and it contrasts with the well documented diversity of penicillin susceptible better document and understand the nature and mechanisms of genetic stability in these ST-2 and ST-3, each with a unique sequence type (MLST) and capsular type have geographic sites of isolation. purpose of this research proposal is to combine the power of whole genome sequencing pneumococci. resistant and non-vaccine type pneumococcal lineages emerged in the in vivo evolutionary changes in the structure of natural populations of this pathogen. environment. achieved pandemic spread and appear to remain stable over long times and distant (WGS) and epidemiology to obtain insights into the mechanisms by which antibiotic Project III. Project II. Project I. R clones. The studies will have three foci of concentration. The purpose of this project is to use whole genome sequencing (WGS) to Three highly penicillin resistant pneumococcal (PRPn) clones: Pneumococcal strains expressing the non-vaccine type (NVT) such as Such genetic stability in a highly recombinogenic pathogen 11A, 6A or 19A have been identified as minority +-' WGS will be used to test if the and because their ST-1, even '"; -0-0 O." 0-0 (/1 .,, ... O-2 Z E 0-0 (gyp f0/1 8)o +L-' I-a) 53' =_i. RevisedSpecific Aim Section 1R01AI108008903953-50-1OAI1AI""IImpactofAntiibiotics and Vaccines onthe invivo Evolution of S. pneumoniae" 00p Below are themodified specific aims we propose to complete within the two year funding period groups of strains; build gene-sharing trees showing the evolutionary relationships among strains and metabolic reconstructions among the isolates'sequences identified in SA-1a and b to the same time;2) isolates of the same clone recovered from multiple children attending on different continents;and 5) isolates recovered over long time periods of the same pandemic clone recovered from various disease sites in different countries clone recovered from children in different DCCs within weeks of one another;4) isolates the same day care center (DCC) at the same time;3) isolates of the same pandemic -U) Perform whole genome alignments, and bioinformatic comparative genomic and the same day care center (DCC) at the same time; clone recovered from children in different DCCs within weeks of one another;4) isolates on different continents;and 5) of the same pandemic clone recovered from various disease sites in the same time;2) isolates of the same clone recovered from multiple children attending constellation of sources including multiple: 1) isolates recovered from the same child at c: isolates recovered over long time periods 1) isolates recovered from the same child at 3) isolates of the same pandemic different countries y.0 SA-1 Perrfforrmwhollegenomesequencing (WGS) and comparative genomic analyses among:: 1)Spcarrriaiageisisolalatetessoffththeeppaannddeemiciclcolnoen,eS, PS1PI(N(N=10);2) Sp carriage isolates the pandemic clones SP2, &SP3 (n's = 4 and 4, respectively), and 3) invasive isolates of SP1I, 2, &3(n = 16) aswell as 8 nonclonal invasive isolates to: a: detetermrminineififththesuprragenomeofftthepandemiciccllonesiis restricted in size compared with the nonclonal clinical Sp isolates (as suggested by ourpreliminary results examining a limited numberofSP-1 isolates) b: by characterizing nasopharyngeal (NP) isolates from pre-school age children (as it is generally agreed that the NP is the primary site of Sp evolutionary change) from a constellation of sources including multiple: --(Q ,-. N-.0 (s] CAN cap 3:r O7y qty frail metabolic reconstructions among the isolates sequences identified in SA-la and b to groups of strains; build gene-sharing trees showing the evolutionary relationships among strains and c: Perform whole genome alignments, and bioinformatic comparative genomic and -[unreadable]O [unreadable]-U C.-.0 (L) 0)0' infection model. the strains are derived from the known pandemic strains;2) if there are novel clones vitro tissue culture assays for adherence and invasion;and 2) the mouse intraperitoneal against themselves and all other sequenced pneumococcal strains to see: emerging;and 3) a: analyze the WGS from SA-1 b: characterize the colonizing and disease potential of these strains by using 1) in L-. which, if any of the other Sp they cluster with evolutionarily. SA-2: Thepaththogenicicppotetentitaialloffpneumococallsttrraininsselelecttedbyttheconjjugate vaccine is unknown. Thesenon--vaciinetype(NVT)straiinscould beeither: a) an expansion of NVTstrains pre-existing as minority isolates at colonization sites;orb) the products of recombination in which "successful" clonal lineages acquired novel NVT capsules. Thepurposeoftheproposed experiments are to a: analyze the WGS from SA-1 from the invasive NVTstraiinsand comparethem against themselves and all other sequenced pneumococcal strains to see: 1) ifany of the strains are derived from the known pandemic strains;2) if there are novel clones emerging;and 3) which, if any of the other Sp they cluster with evolutionarily. b: characterize the colonizing and disease potential of these strains by using 1) in vitro tissue culture assays for adherence and invasion;and 2) the mouse intraperitoneal infection model. acs 4)> 3-p o-[unreadable] O-0 ',[unreadable] mo[unreadable][unreadable]>[unreadable] (Oil SA-3: DetetermrminineeififththeeppeenRstrains: 1) are lless recombinogenicand/orlliimiittedwiith respect to donor DNA source in in vitro transformation experiments;2) are less capable or incapable of forming mixed strain bioflilm structures, and demonstrate lower transformation rates within in vitro biofilms 'off Revised Public Health Relevance Se,d^ction 1R01AI0l080099353-50-1OAI1AI "Impact of Antibiotics and Vaccines on the in vivo Evolution of S. pneumoniae" Narrative to take their place. We will also study the genetics of these replacement types to see if coats (what the vaccine is directed against) or whether these escape strains are truly they possess most of the genes of the clonal lineages that have just hidden in new outer fundamentally alters their cell surface which could limit their interactions with other new with respect to their entire genetics. reducing the incidence of those types, unfortunately other nonvaccine types have arisen introduction a few years ago of a vaccine against some types of Sp was successful in reproductively isolated, or partially isolated, from the rest of the Sp strains. The strains. Therefore, we propose studies to determine if these penR strains have become 'FD Streptococcuspneumoniae (Sp) is a cause of m